Alleviation of symptoms associated with inflammatory disease states

ABSTRACT

Disclosed are methods for the alleviation of symptoms associated with inflammatory disease states, and more particularly to the inhibition of amatory processes associated with the multiple sclerosis disease, by administering a pharmaceutically effective amount of antibody substance immunologically reactive with the common β chain (CD18) of human leukocyte integrins and/or competes with mAb 60.3 for binding to human LPA-1.

BACKGROUND OF THE INVENTION

The-present invention relates generally to methods for the alleviation of symptoms associated with inflammatory disease states, and more particularly to the inhibition of inflammatory processes through administration of a pharmaceutically effective amount of an antibody substance immunologically reactive with molecules expressed on the surface of leukocytes.

Inflammation is a body process central to a number of diseases and is the body's primary defense against infection. The inflammatory process involves an orchestrated series of events initiated in response to tissue damage. In all, cases, this cellular damage ultimately leads to the influx of white blood cells (leukocytes) to the site of injury. As leukocytes arrive at the site of injury, they become metabolically activated and begin to secrete specific proteins (mediators) that are generally of defensive significance, for example, in the eradication of bacteria.

If unregulated, the inflammatory state may persist as a condition known as chronic inflammation. In this setting, the mediators produced may amplify the inflammatory response and cause damage to otherwise normal tissue. Depending upon the body site, such tissue damage may result in chronic diseases such as arthritis, multiple sclerosis, asthma, emphysema, ulcerative colitis, and various autoimmune diseases.

Multiple sclerosis (MS) is a chronic disease characterized by recurrent attacks of neurologic dysfunction due to lesions in the central nervous system. These lesions, termed “plaques,” represent areas of axonal demyelination which are the hallmark of multiple sclerosis. The lesions contain inflammatory cells such as lymphocytes, macrophages and neutrophils in areas where myelin is being destroyed. The classic clinical features of multiple sclerosis include impaired vision and weakness or paralysis of one or more limbs. After a number of years, patients experience a slow, steady deterioration of neurologic function. The disease course is unpredictable and involves exacerbations and remissions in 75% of patients. Although a few patients die within the first few years of onset, the average duration of disease is greater than 30 years.

There are an estimated 250,000 cases of multiple sclerosis in the United States, with approximately 10,000 new cases occurring each year. The cause is unknown but epidemiology implicates immunologic or infectious factors resulting in a chronic inflammatory brain condition. Multiple sclerosis is a disease of young adults, with 66% of cases occurring at ages 20-40; 60% of the patients are women. Over one million physician visits occur annually for multiple sclerosis in the United States alone; however, there is currently no effective treatment-for multiple sclerosis. Therapy is directed toward the reduction of the severity of acute episodes, and prevention of relapses. In acute flare-ups, steroids reduce severity and speed recovery. Experimental therapy with, other immunosuppressive agents, such as cyclophosphamide, has been tried, but with limited success.

A model for human multiple sclerosis is experimental allergic encephalomyelitis (EAE), an acute inflammatory and demyelinating disease of the central nervous system (CNS). [Rose, et al., Clin. Immunol. Immunopathol., 59:1-15 (1991)]. For both EAE and MS, there is considerable evidence that immunological and inflammatory processes, contribute to the pathogenesis of the disease. [Hauser, et al., Ann Neurol 13:418-425 (1983); Traugott, et al., Cell. Immunol., 68:261-275 (1982); Rose, et al., Clin. Immunol. Immunopathol., 45:405-423 (1987). This is supported by the presence of perivascular mononuclear cellular infiltrates in lesions and macrophage-dependent phagocytosis of myelin in the CNS white matter. [Prineas, et al., Lab. Invest., 38:409-421 (1978); Alvord, J C Koetsier, editor; Handbook of Clinical Neurol, 3(47):467-502, Koetsier (ed.); Amsterdam; Elsevier Science Publishers BV (1985)]. The mechanisms by which these blood cells first recognize the brain as a target organ and then traverse the blood brain barrier are not well understood.

The migration of blood cells into extravascular sites of inflammation involves a complex series of events including: i) recognition of an intravascular chemotactic stimulus, ii) adherence to endothelium, iii) diapedesis across the endothelium, and iv) migration through subendothelial connective tissue. [Snyderman, et al. Science, 213:830-837 (1981)]. Endothelial cells (EC) found on lumenal surfaces of blood vessels are the first cells that leukocytes encounter during migration from the blood to the extravascular space. Molecules expressed by both the leukocytes and by the endothcelial cells are important in regulating the adhesive interaction between these two cell types.

One family of leukocyte receptors, variously designated “leukocyte integrins,” “leukointegrins,” and “CD11/CD18 integrins,” are involved in cell-cell and cell-protein interactions of all leukocytes. [Hynes, Cell, 48:549-554 (1987); Beatty, et al., J. Immunol., 131:2913-2918(1983)]. The CD11/CD18 antigen family consists of three heterodimers, each containing a unique α-chain (CD11a, CD11b, or CD11c), and a common β-chain (CD18). [Sanchez-Madrid, et al., J. Exp. Med., 158:1785-1803 (1983)]. The CD11a/CD18 integrin is referred to as LFA-1; the CD11b/CD18 integrin is referred to as Mac-1; the CD11c/CD18 integrin is referred to as p 150,95.

Numerous murine hybridomas have been generated which produce monoclonal antibodies of varying isotype to the common β chain (CD18) of the leukocyte integrins. These include: mAb 1B4 [IgG2a; Wright, et al., Proc. Nat'l. Acad. Sd. USA, 80:5699-5703 (1983)]; mAb 60.3 [IgG2a; Beatty, et al., J. Immunol., 131:2913-2918 (1983)]; mAb TS1/18 [IgG1; Sanchetz-Madrid, supra]; mAb H52 (Hildreth, et al., Science 244:1075-1803 (1989)]; and ATCC T1B 218 [IgG2a Kappa; Springer, et al., J. Exp. Med., 158:586-602 (1983)]. Production of chimeric and humanized monoclonal antibodies against human CD18 is referred to in Law, et al., European Patent Application 440 351 A2, published Aug. 7, 1992.

Monoclonal antibodies directed against the common β-chain of leukocyte integrins completely inhibit adherence of PMNCs to un-activated endothelial cells and certain matrix proteins in vitro [Harlan, et al., Blood, 66:167-178 (1985); Zimmerman, et al., J. Clin. Invest., 81:531-537 (1988); Bohnsack, et al., J. Exp. Med., 171:1221-1237 (1990); Luscinskas, et al., J. Immunol., 142:2257-2263 (1989)]. Moreover, the systemic administration of anti-CD11/CD18 antibodies inhibits, tissue accumulation of PMNCS. [Springer, et al., Nature, 346:425-434 (1990); Jutila, et al. Transplantation, 48:727-731 (1989); Arfors, et al., Blood, 60:338-340 (1987); Price, et al., J. Immunol., 139:4174-4177 (1987); and, Doerschuk, et al., J. Immunol., 144:2327-2333 (1990)].

Arfors, et al., supra, studied the effect of the anti-CD18 antibody, mAb 60.3, on induced PMN accumulation in vivo and found that both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skin lesions of rabbits treated with mAb 60.3 prior to intra dermal injection with the chemotactic factors fMLP, leukotriene (LTB₄) and C5a. These chemotactic agents cause a significant increase in albumin extravasation; extravasation of PMNs at chemotaxin-injective sites is followed by plasma leakage. Curiously, histamine-induced PMN-independent plasma leakage was unaffected by pre-treatment with mAb 60.3.

Hernandez, et al., Am. J. Physiol. 253:H699 (1987) investigated whether PMNs mediate the increase in microvascular permeability produced by ischemia-reperfusion (I/R) by treating cats with either saline, anti-neutrophil serum (ANS), or mAb 60.3. The results indicated that both PMN depletion and prevention of PMN adherence significantly attenuated the increase in microvascular permeability induced by I/R and that prevention of neutrophil adherence with mAb 60.3 afforded protection against I/R-induced microvascular injury. See also, U.S. Pat. No. 4,797,277. Similarly, Vedder, et al., J. Clin. Invest., 81:939 (1988) concluded that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after general ischemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the mAb 60.3. Their results, however, did not rule out the possibility that other leukocytes or other leukocyte adherence functions may also be involved, as CD18 is present on all leukocytes.

Doerschuk, et al., supra, reported on the differential effects of mAb 60.3 on neutrophil (PMN) migration to either pulmonary or peritoneal sites of inflammation in rabbits. Inflammation in the abdominal wall of rabbits was induced following implantation of polyvinyl sponges containing one of the following stimuli: Streptococcus pneumoniae, endotoxin (E. coli), hydrochloric acid or phorbol myristate acetate (PMA). Peritoneal inflammation was induced by instillation of S. pneumoniae and pulmonary inflammation was induced by instilling intrabronchially either S. pneumoniae, hydrochloric acid, E. coli endotoxin, or PMA. Rabbits were pre-treated with either intra venous injection of mAb 60.3 or saline 20 minutes prior to initiating inflammation. The results demonstrated that mAb 60.3 inhibited PMN emigration in response to S. pneumoniae, hydrochloric acid, E. coli endotoxin, or PMA into both the abdominal wall and the peritoneal cavity. However, in the same animals, mAb 60.3 did not alter PMN emigration into the alveolar space, visceral pleura, or bronchial epithelium in response to S. pneumoniae or hydrochloric acid. In contrast, mAb 60.3 markedly inhibited PMN emigration into the alveolar space in response to E. coli endotoxin and abolished lung emigration in response to PMA. The results demonstrated that there, are as yet unidentified organ-specific and stimulus-specific differences in PMN adherence mechanisms that could be due to differences in response of pulmonary and systemic endothelium to identical stimuli or, to the site-specific production of secondary mediators that affect the PM1 or endothelial cell.

In addition to U.S. Pat. No. 4,797,277, which addresses methods for reperfusion therapy with anti-leukocyte-integrin antibody preparations, a number of quite generalized proposals for therapeutic uses of such antibodies have recently been made. PCT WO 92/04034, published Mar. 19, 1992, generically proposes the use of anti-CD18 antibodies in the treatment of endotoxin shock. PCT WO 92/03413, published Mar. 5, 1992, proposes use of CD18 peptide fragments and-antibodies thereto in the treatment of a “disease”. European Patent Application No. 440 451, supra, illustrates usefulness of recombinant humanized anti-CD18 antibodies in the treatment of rabbit dermal inflammation but projects utility in treatment of inflammation in “lung, central nervous system, kidney, joints, endocardium pericardium, eyes, ears, skin, gastrointestinal tract and urogenital systems.” Although multiple sclerosisis proposed as one of many disease states which “may be responsive to recombinant human anti-CD18 antibody,” no studies of the effect of such antibodies on any chronic inflammatory disease states are reported.

There thus continues to exist a need in the art for therapeutic materials and regimens for the treatment of inflammation associated with multiple sclerosis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel and effective methods for the treatment of inflammatory processes and the alleviation of symptoms associated with the multiple sclerosis disease state comprising administering a therapeutically effective amount of an anti-CD18 and/or anti-LFA-1 antibody substance.

Antibody substances useful in practice of the present invention include monoclonal and polyclonal antibodies, antibody fragments, single chain antibodies, chimeric and/or CDR-grafted. (including humanized) antibodies and the like which are specifically immunoreactive with one or more epitopes presented by the common β-chain (CD18) of human leukocyte integrins. Antibodies may be of any class or subclass including IgG, IgA, IgD, IgE and/or IgM. Presently preferred is mouse monoclonal antibody mAB 60.3 of the IgG2a isotype.

Also provided by the present invention are monoclonal anti-human CD18 antibodies produced by hybridoma cell lines designated 23F2G (A.T.C.C. HB 11081) 23I11B and 22F12C. These and antibodies produced by hybridomas 22B3B and 22J4A are characterized as competitive inhibitors of the binding of mAb 60.3 to human LFA-1 on Hut78 cells.

Other aspects and advantages of the present invention will be apparent upon consideration of the enclosed detailed description of an embodiment thereof, reference being had to the drawing wherein:

FIGS. 1 and 2 present magnetic resonance images illustrating results of practice of the invention in the Experimental Allergic Encephalomyelitis (EAE) model system; and

FIGS. 3 and 4 represent histological results of practice of the invention in the EAE model system.

DETAILED DESCRIPTION

The following detailed description illustrates practice of the invention through use of the mouse monoclonal anti-CD18 antibody, mAb 60.3, to alleviate progression of Experimental Allergic Encephalomyelitis (EAE) in macaque monkeys. Non-human primates induced to develop EAE, an acute inflammatory disease of the central nervous system and a model of the human disease MS, were chosen because of the many immunological and neurophysiological similarities EAE shares with MS (Rose, et al., supra.) and because the CD18 antigen recognized by mAb 60.3 is present in high density on monkey peripheral blood leukocytes (Rose, et al., Clin. Immunol. Immunopathol, 44:93-106 (1987). In vivo administration of the antibody at the onset of clinical disease significantly prolonged survival of five of six macaques and in some cases completely reversed clinical appearance of disease.

Example 1 relates to the induction of EAE in monkeys. Example 2 describes treatment by mAb 60.3 antibody infusion and Example 3 relates to the evaluation of disease progression as monitored by a combination of clinical evaluation, blood analysis, and magnetic resonance imaging, including immunocytochemical staining of frozen tissue sections obtained post-mortem. Example 4 describes the preparation of hybridoma cell lines producing anti-human CD18 antibodies and Example 5 relates use of monoclonal antibodies from hybridoma 23F2G in treatment of EAE.

EXAMPLE 1

Induction of EAE

Eighteen male monkeys Macaca fasciculatis weighing 2-3 kg were sensitized to myelin basic protein (BP) by the intra dermal injection of 0.1 ml of an emulsion containing 5.0 mg of monkey BP and 0.5 mg of heat-killed M. tuberculosis. Each animal was obtained and housed by the Regional Primate Research Center at the University of Washington, Seattle. The Primate Center conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ten days after sensitization to BP, each animal was outfitted with a femoral catheter and tether to facilitate blood sampling and the administration of treatments or anesthetics for magnetic resonance imaging (MRI). The animals were maintained on intravenous heparin (18 units/hour) to prevent blood clots from forming in the intra venous lines.

The animals were randomly admitted to specific treatment groups before they were sensitized to BP. This was done to schedule MRI and to prevent the severity of clinical signs at the onset from influencing the choice of therapy. Primate Center personnel, responsible for deciding when animals should be treated or sacrificed, were blinded regarding the treatment history of each animal. Table 1 below provides a statement of criteria for determination of clinical signs and grades of EAE in the animals. TABLE 1 CLINICAL SIGNS AND GRADES OF EAE IN MACAQUES Score Signs ? Prodromal signs, including weight loss, anorexia, yawning, slow response to stimuli, irritability ± Mild neurologic signs, including “headache” (acute distress), “apathy” (indifference), hypokinesia, drooling, clumsiness in using limbs, nystagmus + Moderate neurologic signs, including akinesia, blindness, ataxia, tremor, ptosis, seizures, paresis, incontinence ++ Severe neurologic signs including somnolence, paraplegia, hemiplegia, or quadriplegia +++ Moribund state with semicoma, coma, decerebration or decortication D Death

EXAMPLE 2

Treatment By Antibody Infusion

At the onset of definite clinical signs (± to +) six EAE animals received a bolus injection of mAb 60.3 (2 mg/kg) and dexamethasone (4 mg/kg). Dexamethasone was administered to reduce the incidence of severe acute EAE which is rapidly fatal within 48 hours unless treated immediately. Sever acute EAE occurs in approximately 25% of animals induced to develop EAE and is caused by edema in areas involving or impinging upon the brain stem or cerebral cortex. Following injection of the bolus, these six animals were treated by continuous infusion of mAb 60.3 (2 mg/kg/day) for seven days. Over the seven day treatment period, the dose of dexamethasone, which started at 4 mg/kg, was halved every two days until only 1 mg had been in effect for three days. Six control EAE animals were treated only with dexamethasone, following the same protocol outlined above, and six additional controls were treated with continuous infusion of saline.

Animals that improved and/or stabilized as a result of therapy were sacrificed six weeks after the onset of clinical signs. Animals that experienced a clinical relapse (clinical signs ≧++) some time after the cessation of treatment and before six weeks had elapsed, were sacrificed shortly after the onset of the relapse. Animals that did not respond well to therapy and continued to deteriorate clinically (clinical signs ≧++) were sacrificed without completing the treatment program. The statistical significance of differences in survival times in each treatment group was determined by two-tailed Student t analysis.

EXAMPLE 3

Evaluation of Disease Progression

Disease progression was monitored by a combination of clinical evaluation, blood analysis and magnetic resonance imaging. Clinical evaluations were performed twice daily.

As indicated in Table 2 below, all of the animals in this study developed clinical signs of EAE 13-26 days after sensitization to myelin basic protein.

In Table 2, listing of animal numbers in boldface identified animals scanned by MRI; the Onset/Grade column reflects the day of onset after sensitization and the severity of clinical signs (see Table 1) at onset; the Survival/Grade column reflects days of survival post-onset and severity of clinical signs at time of sacrifice. TABLE 2 THERAPEUTIC RESULTS Animals Onset/Grade Treatment Survival/Grade 89070 17 days/+ 60.3 + dex  2 days/D 89075 21 days/+ 60.3 + dex 42 days/± 89080 21 days/+ 60.3 + dex 42 days/± 89069 18 days/± 60.3 + dex 42 days/± 89071 18 days/± 60.3 + dex 21 days/++ 89074 22 days/+ 60.3 + dex 13 days/+ 87143 21 days/± dex alone 17 days/++ 87125 24 days/± dex alone  1 day/++ 87209 21 days/+ dex alone  2 days/++ 89077 16 days/± dex alone  1 day/D 87200 22 days/+ dex alone 13 days/++ 87069 26 days/± dex alone  9 days/++ 84228 13 days/+ saline  5 days/D 84218 19 days/+ saline  3 days/D 84291 25 days/+ saline  1 day/D 86140 26 days/+ saline  3 days/D 86187 19 days/+ saline  1 day/++ 86209 15 days/+ saline  1 day/++

As indicated in Table 2, all eighteen animals developed clinical signs of EAE which ranged in severity from ± to + at the onset of disease. Six of the eighteen EAE animals (33%) died of severe acute EAE 1-5 days after onset. Four of the six had been treated with saline, one with dexamethasone, and one with a combination of dexamethasone and mAb 60.3. The remaining twelve animals were, sacrificed at various times after, onset either because they had deteriorated clinically or because they had completed the six-week post-treatment protocol.

Out of six mAb-treated animals, five either improved or stabilized with continuing, but not worsening, clinical signs. Animal 89071 improved, but suffered an acute relapse two weeks after the cessation of treatment and was sacrificed at 21 days. Animal 89074 was sacrificed at 13 days, one week after the cessation of treatment, to investigate an abnormal lipid signal detected by magnetic resonance spectroscopy (MRS), a technique being developed for studies of demyelination in the nonhuman primate [Richards et al., New York, Soc. Mag. Reson. Med. 2: p.1043 (1990)]. This animal had improved as a result of therapy and was clinically stable at the time of sacrifice. Animals 89075, 89080, and 89069 were all clinically stable six weeks after onset. The mean survival time of mAb 60.3-treated animals was 30 days. Control animals treated with saline (excluding animal 87220) survived 1-5 days after onset, with a mean survival time of 3 days. Animals treated with dexamethasone survived 1-17 days afteronset, with a mean survival time of 7 days, 3 of 6 dying of their disease 1-2 days after onset, and 3 of 6 surviving 9,13 and 17 days, respectively, until they were sacrificed due to deteriorating clinical signs (≧++).

Magnetic Resonance Imaging

MRI was used to map the anatomic distribution of the lesions in all six of the mAb-treated EAE animals, two dexamethasone-treated animals, and two saline-treated animals. Conventional spin-warp imaging was performed using a General Electric CSI-II. NMR imager/spectrometer (2 Tesla magnet) as described in Rose et al., Biomed. and Pharmacother., 43:347-353 (1989). Images were acquired once a week before the onset of clinical signs and twice a week after the onset of clinical signs. The animals were anesthetized with ketamine and positioned in the magnet so that the center of the brain was at magnet iso-center. Coronal T2-weighted images (TE=80 msec, TR=3000 msec) were used to find abnormal and normal brain regions. Lesions were detected in 60% of the scanned animals on or before the onset of clinical signs. Most lesions were localized in the cerebral white, matter, optic tracts, visual radiations or brainstem. The lesions were identified as areas of increased brightness (white) in a T2-weighted MR image. Of the four control animals that were scanned, only one survived long enough to obtain post-treatment scans. In this dexamethasone-treated animal (87069) a brainstem lesion, detected one day before the onset of clinical signs, progressed in size and intensity until the animal was sacrificed nine days later as shown in FIG. 1. Plate A represents a “normal” brain scan, 21 days after sensitization; Plate B reveals detection of a brainstem lesion two days after onset of clinical signs i.e., 28 days post sensitization: Plates C and D illustrate gradual progression of the brainstem lesion 35 and 38 days after sensitization, respectively.

Following treatment with mAb 60.3, the MRI lesions detected in 5 of 6 animals diminished in both size and intensity. Three animals (89075, 89071, and 89074) had evidence of brainstem lesions at the onset of clinical signs. Although such lesions are frequently fatal if untreated [Shaw et al., Ann. Neurol. 24:738-748 (1988)], these lesions disappeared in all three animals following treatment with mAb 60.3. FIG. 2 demonstrates the progression of the brainstem lesion in animal 89071. In the Figure, Plate A illustrates a normal brain scan 15 days post sensitization; Plate B shows a brainstem lesion at the onset of clinical signs, i.e., 18 days after sensitization; Plates C through F illustrate gradual resolution of the brainstem lesion through antibody treatment as monitored at 22, 25, 29 and 32 days post sensitization; Plate G illustrates reappearance of a lesion in the same region which was accompanied by clinical relapse 39 days post sensitization and 14 days after cessation of treatment. Enlarging lesions are identified as prominent areas of increased intensity (white) on the scans. As noted above, two weeks after the treatment was discontinued, 89071 clinically relapsed and MR images demonstrated a larger, more intense lesion in the brainstem. The relapse was not treated and the animal was sacrificed.

Animal 89080 developed a major hemorrhagic lesion in the lateral geniculate nucleus and striatum. Hemorrhagic lesions are usually fatal and usually do not respond to treatment (Shaw et al., supra) but this lesion almost completely disappeared following administration of mAb 60.3. Animal 89069 had the mildest disease of any of the animals, as determined by MRI, with several small, but intense, sub-cortical white matter lesions that disappeared following treatment.

Two of the longest surviving animals (89080 and 89069) developed a chronic disease characterized by the appearance of new, smaller, MRI-detectable lesions 25 and 22 days, respectively, after the cessation of treatment. These secondary lesions were not as severe as the first lesions and were accompanied by milder clinical signs, which were not treated. Animal 89074 was sacrificed 13 days after the onset of disease, to investigate an abnormal lipid signal detected by MRS. At the time of sacrifice, six days after stopping treatment, his initial lesions were gone, but a new smaller subclinical lesion had appeared on the opposite side of the brain. No new lesions were ever detected in animal 89075 after cessation of treatment.

Cryostat sections of cerebellum or cerebrum were stained with hematoxylin and eosin and by the immunoperoxidase technique in conjunction with mAbs to human leukocyte membrane antigens in order: to analyze the composition of the cellular infiltrates. Immunocytochemical staining with mAb 60.3, which recognizes a CD18 determinant common to all leukocytes, was used to confirm the presence of leukocytes in EAE lesions. A tissue section obtained from the brain of animal 89070 and stained with mAb 60.3 is shown in FIG. 3. In Panel A, at 390× magnification and stained with rabbit anti-mouse IgG, murine IgG coated cells are revealed in the lumen of blood vessels but not in brain tissue; and, in Panel B, at 390× magnification and stained with mAb 60.3, massive infiltration of PMBCs is seen in the exudate and extending out into adjacent white matter.

Animal 89070 was the only mAb-treated animal to die before completion of treatment. Brain tissue taken from this animal was stained with a goat anti-mouse Ig antibody in order to determine whether the treatment mAb had entered the CNS before the animal died. MAb 60.3 reacts with both leukocytes and brain microglia. If this mAb had crossed the blood-brain-barrier during the treatment period, it was expected that a staining pattern similar to that shown in FIG. 3, Panel B might be obtained. Alternatively, if the treatment mAb entered the CNS, bound only to blood leukocytes, the microglia would not have been stained. Instead, murine Ig-coated cells were only detectable in the vessel lumen, and not at all in the perivascular spaces. The absence of any positively stained leukocytes in the lesion stained with goat anti-mouse Ig, suggests that the animal died of an inflammatory condition existing before treatment was initiated and that the treatment antibody was given too late to be effective.

Effect of Treatments on Leukocyte Counts

The hematologic hallmarks of untreated EAE are a progressive leukocytosis and lymphopenia prior to the onset of clinical signs. The leukocytosis can represent as much as a four-fold increase in the number of circulating PMNC and no significant changes in the frequency or absolute numbers of monocytes, eosinophils, or basophils. Following successful treatments of EAE, the absolute numbers of lymphocytes and PMNC return, fairly soon after the initiation of treatment, to pre-sensitization levels. Thus, continuous monitoring of blood leukocytes provides a useful measure of disease progression which complements the clinical evaluation and magnetic resonance imaging.

Blood was drawn on a weekly basis until the onset of clinical disease and then more frequently. One-half ml was sent to the hematology laboratory for WBC and differential counts. The results are set out in Table 3 below providing white blood cell number (×10³) per mm³ whole blood. “NA” indicates that data was not available. A dashed line indicates that the animal was sacrificed at this time. TABLE 3 WBC ANALYSES OF MAB-TREATED EAE ANIMALS ANIMALS: 89070 89075 89080 89069 89071 89074 Day Presens 8.1 8.7 14.0 8.0 7.8 13.3 Onset 14.6 16.2 16.4 9.3 12.3 18.1 Day 2 — 27.1 33.2 22.2 22.6 49.9 Day 4 49.6 53.1 31.6 41.4 72.0 Day 7 61.2 61.7 16.4 90.7 88.6 Day 10 20.7 18.2 20.0 12.8 14.6 Day 14 33.3 9.6 8.2 18.4 13.4 Day 21 8.7 12.9 8.9 37.1 — Day 28 8.7 19.8 6.7 — Day 35 7.9 16.4 6.9 Day 42 NA NA 8.6 WBC ANALYSIS OF CONTROL EAE ANIMALS DEXAMETHASONE CONTROL Animal: 87143 87125 87209 89077 87200 87069 Presens 4.7 6.1 6.7 13.5 8.9 14.1 Onset 9.6 9.0 14.6 32.2 12.4 16.3 Day 2 3.1 — — — 5.4 NA Day 4 3.7 3.6 NA Day 7 3.8 7.0 NA Day 10 8.7 10.7 — Day 14 7.9 — Day 21 — SALINE CONTROL Animal: 84228 84218 84291 86140 86187 86209 Presens 11.0 7.0 13.3 9.7 8.3 9.7 Onset 14.3 9.0 21.9 23.6 14.2 13.5 Day 2 20.2 8.4 22.5 29.0 — — Day 4 30.1 8.0 — — Day 7 — —

In animals treated with mAb 60.3 and dexamethasone, a delayed resolution of the leukocytosis was observed as compared to animals treated with dexamethasone alone as revealed in Table 3. In two of the dexamethasone-treated animals that survived 9 days or longer, the white blood cell counts dropped to pre-sensitization levels two days after the initiation of treatment. In mAb 60.3-treated animals, a pronounced leukocytosis (4-12 fold above presensitization values) was observed which lasted for the entire treatment period. In these animals, the WBC returned to presensitization levels 3-7 days only after the treatment period had ended. Transient episodes of lymphopenia and leukocytosis continued to be observed in the surviving animals, and could be correlated in some cases with episodes of clinical relapse.

Reaction of the treatment mAb with blood leukocytes was confirmed by staining cells with anti-murine Kappa chain mAb (Becton-Dickinson, San Jose, Calif.). Flow cytometric analysis was performed with a FACSCAN Analyzer (Becton Dickinson, San Jose, Calif.). Forward and right-angle scatter gates were set on lymphocytes or large polymorphonuclear granulocytes for analysis of staining patterns. The results for lymphocytes are set out in Table 4 below. TABLE 4 % MURINE Ig-COATED CELLS ANIMALS: Day 89070 89075 89080 89069 89071 89074 Presens 0 0 0 0 0 0 Onset 0 0 0 0 0 0 Day 2 — 96.6 97.7 94.2 96.7 98.2 Day 4 93.8 95.2 91.2 97.0 49.8 Day 7 95.2 99.1 94.9 95.4 27.2 Day 10 0 2.6 11.2 19.6 1.4

The dose of mAb given: to the animals was in all instances sufficient to coat >95% of circulating white blood cells and, as indicated in Table 5, to maintain detectable levels of free antibody in circulation. Data in the Table represent micrograms of circulating, murine IgG per mm³ whole blood. TABLE 5 CIRCULATING MURINE IgG ANIMALS: Day 89070 89075 89080 89069 89071 89074 Presens 0 0 0 0 0 0 Onset 0 0 0 0 0 0 Day 2 — 65 70 15 28 34 Day 4 80 70 65 25 15 Day 7 90 58 4 7 28 Day 10 0 0 0 0 0 After 7 days, the injected mAb was no longer detectable in the circulation. The disappearance of the murine Ig coincided with the appearance of monkey anti-murine Ig antibodies in the circulation of each animal. Neuropathology

To confirm the diagnosis of EAE, the-brains and spinal cords of each BP-sensitized animal were examined postmortem. The animals were euthanized by Nembutal overdose and brains and spinal cords were removed and dissected so that fresh-frozen samples could be obtained before the larger blocks were fixed in 10% neutral formalin. Representative blocks of tissue were embedded in paraffin and sections were stained with hematoxylin and eosin, gallocyanin-Darrow red, luxol fast blue (LFB) combined with periodic acid Schiff (PAS) and hematoxylin and with Holmes' axon stain alone or combined with LFB to differentiate demyelination from necrosis. Frozen sections of mirror-image blocks were stained with Oil Red O (ORO) [Kil, Stain Technology, 47:271, (1972)] to identify lipid-filled macrophages.

All eighteen EAE animals were evaluated histologically postmortem for evidence of EAE. All had microscopic evidence of either mild to severe acute EAE or hyperacute EAE that generally correlated with the severity and duration of the disease. All but 2 of the 12 control animals showed either predominantly hyperacute lesions characterized by hemorrhages, subtotal necrosis, demyelination, and diffuse massive infiltrations of neutrophils into the CNS parenchyma; or severe acute perivascular lesions which were more compact, more confined to the perivascular spaces than the hyperacute lesions and composed predominantly of lymphocytes and macrophages, but some neutrophils, and with abundant demyelination as well as some axonal debris. The two exceptional control monkeys, with EAE of 13-17 days duration, had a mixture of lesions ranging from mild to severe demyelinating lesions, composed predominantly of lymphocytes and macrophages with abundant myelin debris and little or no axonal reaction. All but one of the mAb-treated monkeys had a combination of: (a) older well developed demyelinated plaques with abundant myelin debris, sudanophilic lipid and little or no axonal reaction or lymphocytic infiltrate; and (b) more recent lesions corresponding to the terminal untreated relapses, which were compact, confined to perivascular spaces, with varying proportions of lymphocytes, monocytes, or neutrophils (consistent with diagnoses of mild to severe acute EAE) and some myelin debris. These two types of lesions, found in the same animal (#89069), are shown in FIG. 4.

In FIG. 4, Panel A, at two-fold magnification, numerous inflammatory (area 1) and/or demyelinating (area 2) lesions can be ascertained in the white matter; in Panel B, at 325× magnification, area 2 of Panel A is revealed as being composed predominantly of macrophages scattered diffusely through the tissue and about blood vessels, with the small black granules constituting PAS positive glyco-lipoproteins; and in Panels C and D, at 130× and 325× magnification, respectively, area 1 of Panel A is revealed as being composed predominantly of PMNCs and no PAS positive insoluble glycoproteins.

Summary of Results

Monitoring disease progression by a combination of clinical evaluation, blood cell fluctuations, and magnetic resonance imaging (MRI) revealed the following. Antibody treated animals survived significantly longer than either dexamethasone- or saline-treated animals (p<0.02; p<0.001; respectively). The effectiveness of this therapy is further confirmed by visualization by magnetic resonance imaging (MRI). The almost complete disappearance of major lesions in the brainstems of three different animals was unprecedented as brainstem lesions are rapidly fatal in untreated animals. The disappearance of MRI-detectable lesions following administration of mAb 60.3 demonstrates a potent effect on the inflammatory reactions producing edema in the brain lesions. MAb 60.3 is known to inhibit plasma leakage in vivo [Arfors et al, Blood, 60: 338-340, (1987)], probably the most important factor in improvement in MRI-detectable lesions, as changes in the tissue water characteristics can be visualized by changes in image intensity on the MRI scans.

The severity of clinical signs at the onset of EAE was not uniform because individual monkeys responded differently to sensitization with myelin BP. Nevertheless, there was no correlation between the severity of clinical signs at the onset of EAE and the ability of animals to respond to treatment. Of the six mAB-treated animals, four were + at the onset of clinical signs and 2 were ±. Of the twelve control animals (six dexamethasone-treated and six saline-treated), 8 were + and 4 were ±, essentially the same distribution as the mAb-treated group. No correlation was found between the severity of clinical signs at the onset and the potentially stressful procedure of MRI examination under general anesthesia. Of the ten animals scanned by MRI, six were + at the onset of clinical disease and four were ±. One might have expected the additional handling involved in scanning the animals, as well as the frequent administration of anesthetics, to result in a generalized suppression of the immune response. [Wall et al., Lab Animals, 19:138-144 (1985), Thomas et al., Clin. Exp. Immunol., 47:457-466 (1982)]. However, the results do not support this hypothesis.

The dexamethasone-treated animals did have longer mean survival time (7 days) than the saline-treated controls (3 days), but the difference was not statistically significant (p<0.1). Thus, although dexamethasone slowed down the disease process, it had little apparent effect on the final outcome of the disease. Co-administration of mAb 60.3, on the other hand, significantly prolonged survival and in some cases completely reversed the clinical and MRI appearances of EAE.

EXAMPLE 4

Six to twelve week old Balb/c mice (Charles River Biotechnical Services, Inc., Wilmington, Mass., IACUC #901103) were immunized with human T cell line Hut 78 to generate anti-CD18 antibodies. For each of two hybridoma-generating fusions (designated fusions 22 and 23) two Balb/c mice were bled retro-orbitally for the collection of pre-immune serum on day 0. On day 2,each animal received a total of 5×10⁶ Hut 78 cells in 0.2 ml sterile PBS intravenously. The mice were then immunized at two week intervals for six weeks and then at monthly intervals for three months. The final monthly boost was of glutaraldehyde fixed Hut 78 cells. Cells were fixed by mixing equal volumes of cells (2×10⁷) and glutaraldehyde (0.1% so that the final concentration of the fixative was equal to 0.05%. The mixture was incubated with swirling for thirty seconds and an equal volume of 0.2M glycine (in PBS) was added to block the reaction. The reaction mixture was washed once with 0.2M glycine (in PBS) and twice with PBS. Cells were resuspended in: PBS at a concentration of 5×10⁶ cells per 0.2 ml PBS. Immune serum was collected via retro-orbital bleeding on day 56 and tested by FACS to determine its reactivity with the immunogen (Hut 78 cells). Three days prior to the projected fusion date, the animals were immunized by intravenous injection with a final dose of 5×10⁶ glutaraldehyde fixed cells.

For each fusion, the spleen from the mouse displaying the highest immune serum titer against the immunogen was removed sterilely. A single-cell suspension was formed by grinding the spleen between the frosted ends of two glass microscope slides submerged in serum free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI)(Gibco, Canada). The cell suspension was filtered through sterile 70-mesh Nitex cell strainer (Becton Dickinson, Parsippany, N.J.), and washed twice by centrifuging at 200 g for 5 minutes and resuspending the pellet in 20 ml serum free RPMI. Thymocytes taken from 3 naive Balb/c mice were prepared in a similar manner.

NS-1 myeloma cells, kept in log phase in RPMI with 11% fetal bovine serum (FBS)(Hyclone Laboratories, Inc., Logan, Utah.) for three days prior to fusion, were centrifuged at 200 g for 5 minutes, and the pellet was washed twice as described in the foregoing paragraph. After washing, each cell suspension brought to a final volume of 10 ml in serum free RPMI, and 10 μl was diluted 1:100. 20 μl of each dilution was removed, mixed with 20 μl 0.4% trypan blue stain in 0.85% saline (Gibco), loaded onto a hemacytometer (Baxter Healthcare Corp., Deerfield, Ill.) and counted.

For each fusion, 2×10⁸ spleen cells were combined with 4×10⁷ NS-1 cells, centrifuged and the supernatant was aspirated the cell pellet was dislodged by tapping the tube and 2 ml of 37° C. PEG 1500 (50% in 75 mM Hepes, pH 8.0)(Boehringer Mannheim) was added with stirring over the course of 1 minute, followed by adding 14 ml of serum free RPMI over 7 minutes. An additional 16 ml RPMI was added and the cells were centrifuged at 200 g for 10 minutes. After discarding the supernatant, the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ thymocytes/ml. The suspension was dispensed into ten 96-well flat bottom tissue culture plates (Corning, United Kingdom) at 200 μl/well. Cells in plates were fed on days 2, 4 and 6 days post fusion by aspirating approximately 100 μl/well plating medium described above except containing 10 units/ml IL-6 and lacking thymocytes.

On day 10 culture supernatants were taken from each well, pooled by column or row and analyzed for the presence of an antibody capable of competing with an FITC-60.3 conjugate (prepared according to Goding, in “Monoclonal Antibodies: Principles and Practice,” 2d Ed., p. 241-280, Academic Press, New York, 1986) for binding to Hut78 cells. The competition assay was performed as follows. Twenty-five μl of a cell suspension containing 10×10⁶ Hut78 cells/ml was combined with 25 μl of a substantial dose of FITC-60.3 (×2560≅0.8 μg/ml) and 50 ml μl of fusion supernatant and incubated for 30 min at 4° C., washed by adding culture media to the cell mixture and centrifuging at 18000 rpm for three minutes. The supernatant was removed by aspiration and the pellet was washed twice with flow cytometric analysis (FACS) medium containing RPMI 2% PBS and 0.1% NaN₃. The reaction mixture was fixed by addition of 1% paraformaldehyde (pH 7.2 in PBS) and transferred to polystyrene tubes for FACS using a Becton Dickenson FACscan analyzer. Of the 960 hybridomas generated in fusion 22, the supernatant of hybridoma No. 22F12C inhibited binding of FITC-60.3 by about 90%; supernatants from hybridoma Nos. 22J4A and 22B3B inhibited binding by about 40% and 10% respectively. Two hybridomas from fusion 23, 23F2G and 23I11B, produced antibodies which inhibited FITC-60.3 binding by ≧95%. These five hybridomas were cloned. Hybridoma cell line 23F2G was deposited with the American Type Culture Collection on Jun. 30, 1992 and accorded accession number A.T.C.C. HB 11081. The isotype of the antibody produced by hybridoma cell line 23F2G (referred to herein as mAb 23F2G) was determined to be IgG2a. Hybridomas 22F12C and 23I11B also produced antibodies of the IgG2a isotype, while hybridomas 22B3B and 22J4A produced IgG1 antibodies.

Antibodies from hybridomas 22F12C, 23F2G and 23I11B were all found, in varying degrees, to: (1) block adhesion of human T cells to activated HUVEC monolayers; (2) block aggregation of 13-acetate (PMA) activated Con A blast cells phorbol 12-myristate; and (3) induce demargination of white blood cells in rabbits. The ability of the IgG1 antibodies produced by hybridomas 22B3B and 22J4A to block adhesion is as yet undetermined; mAb 22J4A was able to block aggregation, but mAb 22B3B was not; both antibodies induced demargination, but to a lesser degree than the others or mAb 60.3. Based on the screening results noted above, it appears likely that, notwithstanding the ability to compete for binding of mAb 60.3 to LPA-1 on Hut 78 cells, mAb 22B3B and 22J4A are specific for an epitope present on the CD11a component of LFA-1 or an epitope associated with the CD11a/CD18 heterodimer rather than with an epitope of CD18.

A comparison was made between the amino acid sequence of the variable region of mAb 60.3 presented by L. Harris at San Francisco, Calif. on May 26-27, 1992 in a poster presented at a conference entitled “Cell Adhesion Molecules In Inflammation” organized by International Business Communications (South Natick, Mass.) and the amino acid sequence of the variable region of mAb 23F2G. Gross differences were found, despite the fact that 23F2G competed with 60.3 for binding to Hut78 cells. While these differences are suggestive of the possibility that the antibodies bind adjacent, different epitopes on CD18, confirmation of this possibility will have to await definitive epitope-mapping studies.

EXAMPLE 5

Hybridoma 23F2G was amplified by the ascites method and purified by affinity chromatography on Protein A under pyrogen-free conditions. From 25ml of ascites, 120 mg of antibody preparation was isolated. This preparation contained only about 4 endotoxin units per mg.

Two Macaca fascicularis monkeys were then involved in a study to preliminarily determine the ability of mAb ?23F2G to alleviate EAB symptoms. The EAE induction procedures were essentially as in Example 1. Two animals, Nos. 89161 and 89186, sensitized to 1.0 mg of purified monkey basic protein and developed hyperacute hemorrhagic EAE 18 and 20 days post sensitization, respectively. Animal 89161 died within 12 hours of onset and before treatment with dexamethasone could be initiated. Animal 89186 was treated for a week with both dexamethasone (3 days at 4 mg/kg, 2 days at 2 mg/kg and 2. days at 1 mg/kg) and mAb 23F2G (7 days at 2 mg/kg, i.v.). The animal improved dramatically with complete resolution of paralysis three days after initiation of treatment. Lesions detected by MRI resolved four days after cessation of treatment and the animal improved dramatically with complete resolution of paralysis. Eight days following cessation of treatment, the animal relapsed and was sacrificed. Hematologic analysis revealed that mAb 23F2G caused steady demargination of leukocytes which peaked on the last day of treatment. The white blood cell count rose from 22.7×10³ on the day of clinical onset to 82×10³/mm³ on the last day of treatment. The antibody showed saturation of blood lymphocytes at the dose administered.

The foregoing examples describe a method for treatment for inhibiting inflammatory processes and alleviating symptoms associated with inflammatory disease states by administering a reagent, such as an antibody, that blocks leukocyte adhesion and migration to inflammatory sites. While the present invention has been described in terms of specific methods and compositions, it is understood that variations and modifications wit occur to those skilled in the art upon consideration of the description of the present invention. As one example, while two anti-CD18 mAbs, 60.3 and 23F2G, have been demonstrated to be useful in practice of methods of the invention, it is expected that any antibody which competes with mAb 60.3 and/or with 23F2G for binding to LFA-1 will also be useful, whether or not such antibody recognizes on epitope present on CD18. As another example, while mAb 23F2G is a mouse monoclonal antibody, recombinant human antibody preparations comprising the murine complimentarily determining regions of the antibody are being prepared and are expected to be equally suitable for practice of methods of the invention. Accordingly, it is intended in the appended claims to cover all such equivalent variations which come within the scope of the invention as claimed. 

1. A method for inhibition of inflammatory processes and alleviation of symptoms associated with the multiple sclerosis disease state comprising administering a therapeutically effective amount of an anti-CD18 integrin-antibody substance.
 2. The method of claim 1 wherein said anti-CD18 antibody is mAb 60.3.
 3. The method of claim 1 wherein said anti-CD18 antibody is mAb 23F2G.
 4. The method of claim 1 wherein said anti-CD18 antibody is a recombinant human antibody comprising a murine complimentarily determining region of mAb 23F2G.
 5. Hybridoma cell line 23F2G, A.T.C.C. HB11081.
 6. Monoclonal antibody 23F2G produced by hybridoma cell line 23F2G, A.T.C.C. HB
 11081. 7. A method for inhibition of inflammatory processes and alleviation of symptoms associated with the multiple sclerosis disease state comprising administering a therapeutically effective amount of monoclonal antibody which competes with mAb 60.3 for binding to LFA-1. 